Discrimination method of target base in DNA, and allele specific primer used in the method of the same

ABSTRACT

An object of the present invention is to provide an allele specific primer which is accompanied by less possibility of the false positive and enables definite discrimination when a base immediately adjacent to on the 3′ side of a target SNP base is A, while a base adjacent with one base spaced apart is A. According to the present invention, the 3′ end base is designed to be the base corresponding to SNP; the second base from the 3′ end to be C; the third base from the 3′ end to be any one of A or C; and the base sequence of from the fourth from the 3′ end to the 5′ end base to be completely complementary to the sequence of from a base three bases away from the target SNP base on the 3′ side to a desired base.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of discriminating a targetbase carried by a DNA and an allele specific primer for use in the same.

2. Description of the Related Art

SNP is a polymorphism which occurs most frequently among genepolymorphisms, and is believed to appear in human genomes with anincidence of about 0.1%.

As a matter of fact, presence of SNPs of as many as over three millionhas been hitherto elucidated, suggesting that SNP may be very useful asa marker for genetic tests.

Investigations on relationship between SNPs and diseases carried out sofar have revealed close relationship between diseases such as diabetesand hypertension, and SNP.

Known techniques for carrying out SNP discrimination include techniquesin which a primer extension reaction (including PCR reaction) by anallele specific primer is utilized. A primer which yields a markeddifference in efficiency of the primer extension reactions depending onthe base type at targeted SNP is referred to as the allele specificprimer. Therefore, the base type at the target SNP can be readilyspecified, for example, by carrying out a PCR reaction using an allelespecific primer, and analyzing the amount of the PCR product.

In general, allele specific primers are simple origo DNAs which are notanyhow modified especially.

For analysis of the amount of PCR product, so called generalelectrophoretic method may be used. Thus, SNP discrimination techniqueswith an allele specific primer may be referred to as being extremelyadvantageous in terms of cost, reaction time, convenience of operationand the like.

With respect to methods of analysis, in addition to the aforementionedelectrophoretic method, detection by QCM or SPR is also enabled when asolid phase reaction is carried out.

More recently, methods of detecting PPi (pyrophosphate) that is abyproduct of a primer extension reaction utilizing a luciferase reactionhave been also developed. Accordingly, approaches to simplification andacceleration of SNP discrimination with an allele specific primer havebeen elaborately attempted all over the world.

Sequence design of an allele specific primer is a very important factorfor its ability to discriminate SNP.

In the most classical example, 3′ end base corresponds to the target SNPbase (i.e., complementary to any one of predicted base types at thetarget SNP), and other sequence is completely complementary to thetarget DNA sequence. However, in this case, problems of the falsepositive may be raised unless reaction conditions are strictly definedsuch as the reaction time and temperature, or concentration of dNTPsused in the reaction, as well as cycle number when a PCR method isemployed. In other words, fundamentally, although a primer extensionreaction must not be caused from a base corresponding to SNP of theprimer 3′ end when it is noncomplementary to the target SNP base of asample, the reaction is caused in many cases.

In order to solve the aforementioned problems in connection with thefalse positive, several novel allele specific primers have beendeveloped so far.

An allele specific primer proposed in US Patent Publication No.2003/0049628 is a developed allele specific primer in which the 3′ endbase is a base corresponding to SNP, and the third base from the 3′ endwas intentionally made noncomplementary to a base adjacent to on the 3′side of the target SNP base with two bases apart.

US Patent Publication No. 2003/0148301 proposes an allele specificprimer in which the second base from the 3′ end is a base correspondingto SNP, and the third base from the 3′ end base is intentionally madenoncomplementary to a base adjacent to on the 3′ side of the target SNPbase. In this instance, KOD polymerase that is 3′→5′ exo+polymerase ischaracteristically utilized.

US Patent Publication No. 2004/0197803 proposes an allele specificprimer in which 3′ end base is a base corresponding to SNP, and thesecond and the third bases from the 3′ end are intentionally madenoncomplementary to a base adjacent to on the 3′ side of the target SNPbase and a base adjacent with one base spaced apart, respectively. Amongthem, the allele specific primer proposed in US Patent PublicationNo.2004/0197803 involves low possibility of the false positive, inparticular.

More specifically, the allele specific primer disclosed in US PatentPublication No. 2004/0197803 which was designed as described above mayconstruct a loop structure, as shown in FIGS. 1A and 1B, because onlythe second and the third bases from the 3′ end are noncomplementary tothe target DNA sequence (in the Figure, noncomplementary base pair isdenoted by “x”) when the base corresponding to SNP positioned to its 3′end (in the Figure, denoted by “S′”) is complementary to the target SNPbase (in the Figure, denoted by “S′”) (FIG. 1A). Consequently, thepolymerase is efficiently bound to cause a primer extension reaction.

To the contrary, when the base corresponding to SNP positioned to its 3′end is noncomplementary to the target SNP base, all three bases at the3′ end become noncomplementary, and branched structure may beconstructed (FIG. 1B). In this case, the primer extension reaction ishardly caused because binding efficiency of polymerase is believed to beextremely low. Therefore, the allele specific primer proposed in USPatent Publication No. 2004/0197803 shall involve extremely lowpossibility of the false positive as described above.

In addition, the following documents can be referred to as relevantdocuments to the present invention.

Japanese Patent Provisional Publication No. 2004-248635 discloses a PCRprimer for identification of rice variety in which 3′ end corresponds tothe SNP site to be discriminated, and the third base from the 3′ end issubstituted from a sequence that is complementary to the templatesequence to be annealed, with the substitution being G to A, A to C, Tto G, or C to A.

Further, paragraph No. 0040 of Japanese Patent Provisional PublicationNo. 2004-248635 discloses that “In one aspect, the present inventionprovides a primer in which the first, the second, the third or thefourth base from the 3′ end was substituted. This substitution may bejust alone, or a combination of two or more. Preferably, only the thirdbase from the 3′ end is substituted. This substitution of the primer maybe any arbitrary substitution, but is preferably a substitution of G toT, A to C, T to G, or C to A.”.

SUMMARY OF THE INVENTION

The allele specific primer proposed in US Patent Publication No.2004/0197803 exhibits extremely inferior efficiency of the primerextension reaction when the base corresponding to SNP positioned at its3′ end is noncomplementary to the target SNP base. Consequently,problems of the false positive may be resolved, thereby enablingaccurate discrimination of SNP discrimination.

However, efficiency of the primer extension reaction in case where thebase corresponding to SNP positioned at its 3′ end is complementary tothe target SNP base may vary depending on the second and the third basetypes from the 3′ end, and the base type requirements of the target DNAcorresponding thereto (more specifically, base type requirements of abase adjacent to on the 3′ side of the target SNP base and a baseadjacent with one base spaced apart).

More specifically, for example, when a base type at the target SNP inthe target DNA sequence is A (adenine) or G (guanine), and a base typeimmediately adjacent to on the 3′ side of the SNP base is C (cytosine),while a base type adjacent with one base spaced apart is T (thymine), 3′end base of the allele specific primer proposed in US Patent PublicationNo. 2004/0197803 shall be a base corresponding to SNP, and thus may be T(to be complementary to A) or C (to be complementary to G). On the otherhand, the second and the third bases from its 3′ end must benoncomplementary to the target DNA sequence, the second base should beany one of A, T or C, and the third base should be any one of T, G or C.Therefore, there exist 9 kinds of combination in total of the allelespecific primer proposed in US Patent Publication No. 2004/0197803 withrespect to the second and the third base types from its 3′ end for onekind of target DNA sequence. From the perspective that any one isnoncomplementary, these 9 kinds of allele specific primers are supposedto exhibit approximately the same efficiency upon SNP discrimination.

However, the present inventors found that certain allele specificprimers among these 9 kinds of allele specific primers exhibit extremelyhigh efficiency upon SNP discrimination in comparison with other allelespecific primers. Thus, the present invention was accomplished.

Accordingly, an object of the present invention is to provide a methodof discriminating a target base carried by a DNA which is accompanied byless possibility of the false positive and enables definitediscrimination, and an allele specific primer for use in the same.

The present invention provides a method of discriminating a target basecarried by a DNA, the method which comprises:

(1) a DNA elongation step comprising binding to the DNA an allelespecific primer having at its 3′ end a base that is complementary to abase predicted as the target base among 4 bases of A, T, G and C tocause a DNA elongation reaction, and

(2) a discrimination step comprising examining efficiency of the DNAelongation to discriminate that the target is the same as the predictedbase when the efficiency is high, or that the target base is distinctfrom the predicted base when the efficiency is low, wherein

in the DNA, a base immediately adjacent to on the 3′ end side of thetarget base is A, and a base adjacent with one base spaced apart is A,

3′ end base of the allele specific primer is complementary to the basepredicted to be the target base,

the second base from the 3′ end of the allele specific primer is C,

the third base from the 3′ end of the allele specific primer is any oneof A or C, and

a base sequence between the fourth base from the 3′ end and the 5′ endbase of the allele specific primer complementarily binds to a basesequence between the base on the DNA corresponding to the fourth basefrom the 3′ end of the allele specific primer and the base on the DNAcorresponding to the 5′ end base of the allele specific primer.

The DNA elongation reaction is preferably a primer extension reaction.The DNA elongation reaction is preferably a primer extension reactionrelised solely on said allele specific primer. In this case, theefficiency is preferably examined by determining the concentration ofpyrophosphate produced by the primer extension reaction. Further, theconcentration of pyrophosphate is preferably detected in terms ofluminescence intensity.

It is also preferred that the DNA elongation reaction is a PCR reaction.More preferably, the DNA elongation reaction is a PCR reaction relied ona combination of said allele specific primer with the other differentprimer. In this case, the efficiency is preferably examined by measuringthe concentration of the amplified DNA produced by the PCR reaction withan electrophoretic method.

The allele specific primer which may be used in the above method is alsoinvolved in an aspect of the present invention.

According to the present invention, a method of discriminating a targetbase carried by a DNA which is accompanied by less possibility of thefalse positive and enables definite discrimination of SNP is provided.

The foregoing object, other object, features and advantages of thepresent invention will be apparent from the following detaileddescription of the preferred embodiments with reference to theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic view illustrating a relationship between atemplate DNA 2 and an allele specific primer 1 according to conventionalarts.

FIG. 1B is a schematic view illustrating a relationship between atemplate DNA 2 and an allele specific primer 1 FIG. 2A is a schematicview illustrating a relationship between a template DNA 4 and an allelespecific primer 3 according to the present invention.

FIG. 2B is a schematic view illustrating a relationship between atemplate DNA 4 and an allele specific primer 3 according to the presentinvention.

FIG. 3 is a flow chart of a method of discriminating SNP in Embodiment2.

FIG. 4 is a flow chart of a method of discriminating SNP in Embodiment3.

FIG. 5 is a graph presenting experimental results in Example 1.

FIG. 6 is a graph presenting experimental results in Comparative Example1.

FIG. 7 is a graph presenting experimental results in Example 2.

FIG. 8 is a graph presenting experimental results in Comparative Example2.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, embodiments of the present invention will be explained withreference to FIG. 2A to FIG. 4.

Embodiment 1

In this Embodiment 1, base type requirements for the allele specificprimer according to the present invention will be explained withreference to FIGS. 2A and 2B.

In the present invention, as shown in FIGS. 2A and 2B, the target DNAsequence to be a subject includes the target SNP base present at the 5′end, the second base from the 5′ end being A and the third base from the5′ end being A.

In order to facilitate understanding of the present invention, it isassumed that the target SNP base is present at the 5′ end in FIGS. 2Aand 2B. However, in general, the target SNP base may be present anywherein the target DNA sequence, as shown in FIGS. 1A and 1B, and it is notnecessary to exist at the 5′ end of the target DNA sequence.

Because the 3′ end base of the allele specific primer is a basecorresponding to SNP, in case where there are two kinds of predictedbase type at the target SNP (denoted as S1, S2 in FIGS. 2A and 2B), the3′ end base of the allele specific primer is designed such thatcomplementarity is provided to either one of them. In FIGS. 2A and 1B,this base corresponding to SNP is denoted as S1′ which is complementaryto S1.

Moreover, the second base from the 3′ end of the allele specific primeris C, and the third base from the 3′ end is any one of A or C.

Base sequence between the fourth base from the 3′ end and the 5′ endbase of the allele specific primer (hereinafter, may be referred to as“residual base sequence” or “residual base sequence of allele specificprimer”) is complementary and binds to the base sequence between thebase on the target DNA corresponding to the fourth base from the 3′ endof the allele specific primer and the base on the target DNAcorresponding to the 5′ end base of the allele specific primer(hereinafter, may be referred to as “residual base sequence” or“residual base sequence on the target DNA”).

In FIGS. 2A and 2B, the base sequence of from the fourth base from the3′ end to the 5′ end base of the allele specific primer is complementaryto the base sequence of from the fourth base from the 5′ end to the 3′end base in the target DNA sequence.

It is desired that the residual base sequence of the allele specificprimer and the residual base sequence of the target DNA are completelycomplementary, but it is not necessarily completely complementary aslong as any adverse effect is not exerted on the SNP discrimination.

According to the present invention, when the base corresponding to SNPof the allele specific primer is complementary to the target SNP base,relationship as shown in FIG. 2A is provided. To the contrary, when thebase corresponding to SNP of the allele specific primer isnoncomplementary to the target SNP base, relationship as shown in FIG.2B is provided.

In FIGS. 2A and 2B (and FIGS. 1A and 1B), the reference sign “X” meansthat the two bases sandwiching the reference sign X therebetween are notbound. The two bases without the reference sign “X” sandwichedtherebetween (for example, the base S1 and the base S1′ in FIG. 1A andFIG. 2A) are bound.

When a DNA elongation reaction (i.e., typically a primer extensionreaction or a PCR reaction) is carried out with the sequencerequirements of such an allele specific primer and a target DNA usingpolymerase without 3′→5′ exonuclease activity, the reaction proceedsvery efficiently in case of FIG. 2A, while the reaction hardly proceedsin case of FIG. 2B.

The target SNP base carried by the DNA can be discriminated based onsuch difference in efficiency. More specifically, upon examination ofthe efficiency in the DNA elongation step, high efficiency leads todiscrimination that the target SNP base is identical with the predictedbase, while low efficiency leads to discrimination that the target SNPbase is different from the predicted base.

Consequently, SNP discrimination is enabled which is accompanied byextremely low possibility of the false positive and is definite.

Embodiment 2

In this Embodiment 2, a method of discriminating SNP by carrying out aprimer extension reaction using an allele specific primer according tothe present invention will be explained.

As shown in FIG. 3, a double stranded DNA including a target DNAsequence is first prepared so that a primer extension reaction can becarried out. In the target DNA sequence requirements, the target SNPbase is present at the 5′ end, with the second base from the 5′ endbeing A and the third base from the 5′ end being A, similarly toEmbodiment 1.

Therefore, the complementary chain sequence of the target DNA sequencehas its 3′ end base being complementary to the target SNP base, with thesecond base from the 3′ end being T and the third base from the 3′ endbeing T.

The double stranded DNA that meets such sequence requirements isprepared to provide the state to permit the primer extension reaction tobe carried out as described above. Thus, a primer extension reaction maybe carried out through adding the allele specific primer that meets thesequence requirements demonstrated in Embodiment 1, DNA polymerase,dNTPs, a buffer, and a desired salt as needed.

Step of the primer extension reaction may be conducted by any knownprocedure, which may be specifically, as follows.

That is, the aforementioned double stranded DNA is first stood under ahigh temperature condition for allowing denaturation, followed bylowering of the temperature such that binding of the allele specificprimer according to the present invention with the target DNA sequenceis permitted. Thereafter, the temperature condition may be set to allowthe DNA polymerase to execute an elongation reaction.

Consequently, when the base corresponding to SNP positioned at the 3′end of the allele specific primer is complementary to the target SNPbase, the primer extension reaction with this allele specific primer canbe efficiently perfected. To the contrary, when the base correspondingto SNP positioned at the 3′ end of the allele specific primer isnoncomplementary to the target SNP base, efficiency of the primerextension reaction with this allele specific primer is significantlypoor. Therefore, efficiency of the primer extension reaction may beexamined during or following this primer extension reaction by measuringthe amount of pyrophosphate released as a result of the primer extensionreaction, or the like. Accordingly, SNP discrimination is enabled whichis accompanied by less possibility of the false positive and isdefinite.

Procedure for examining efficiency of the primer extension reaction isnot limited to the method of measuring the amount of pyrophosphate.

For example, efficiency of the primer extension reaction may be examinedby carrying out the aforementioned primer extension reaction in thestate in which a 5′ end area of the allele specific primer according tothe present invention is immobilized on a crystal oscillator, andmonitoring the primer extension reaction through analyzing change in theoscillation frequency.

Alternatively, efficiency of the primer extension reaction may beexamined by carrying out the aforementioned primer extension reaction inthe state in which a 5′ end area of the allele specific primer accordingto the present invention is immobilized on a gold surface, andmonitoring the primer extension reaction through analyzing the surfaceplasmon resonance (SPR) phenomenon.

Any procedure is permitted as long as efficiency of the primer extensionreaction can be examined.

Specific procedure for carrying out the primer extension reaction is notlimited to those described above. More specifically, all necessaryreagents are previously mixed into a reaction mixture to execute theprimer extension reaction in the procedure described above, however, forexample, a buffer solution in which the double stranded DNA including atarget DNA sequence and the allele specific primer dissolved therein (adesired salt may be added as needed) may be prepared first, and then adenaturation step at a high temperature and binding to the target DNAsequence with the allele specific primer may be allowed, followed byserially adding DNA polymerase and dNTPs thereto to execute thereaction. Thus, any step may be permitted as long as the primerextension reaction can be perfected.

Also, in case where the DNA including a target DNA sequence isoriginally single stranded, step of standing under a high temperaturecondition intending to allow the double stranded DNA to be denatured isnot required unlike in the case of the double stranded DNA sequence.

However, the single stranded DNA and the allele specific primer must beconditioned to provide a state in which both of them are sufficientlydissociated intending to control their binding reaction. Therefore, itis preferred that a step of standing them under a high temperaturecondition is conducted prior to the binding step of the allele specificprimer and the target DNA sequence.

The DNA polymerase herein preferably has week or no 3′→5′ exonucleaseactivity, which may result in more accurate SNP discrimination.Furthermore, the DNA polymerase can be exposed to a high temperaturedepending on the procedure of the primer extension reaction as describedabove. In such a case, heat resistance enzyme must be used such as e.g.,one for use in PCR, however, it is not particularly necessary in casewhere the primer extension reaction is carried out by a procedure toavoid exposure of the enzyme to such a high temperature.

Examples of the method of measuring the amount of pyrophosphate includethose proposed in Nucleic Acids Research, 2001, Vol. 29, No. 19 e93, WO03/078655 A1, Japanese Patent Provisional Publication No. 2004-141158and the like. However, the method is not limited thereto, but any methodcapable of measuring pyrophosphate is permitted.

Embodiment 3

In this Embodiment 3, a method of discriminating SNP by carrying out PCRusing the allele specific primer according to the present invention willbe explained.

As shown in FIG. 4, a double stranded DNA including a target DNAsequence is first prepared so that a PCR reaction can be carried out. Inthe target DNA sequence requirements then, the target SNP base ispresent at the 5′ end, with the second base from the 5′ end being A andthe third base from the 5′ end being A, similarly to Embodiment 1.

Therefore, the complementary chain sequence of the target DNA sequencehas its 3′ end base being complementary to the target SNP base, with thesecond base from the 3′ end being T and the third base from the 3′ endbeing T.

The double stranded DNA that meets such sequence requirements isprepared to give the state to permit the PCR reaction to be carried outas described above. Thus, a PCR reaction may be carried out throughadding the allele specific primer that meets the sequence requirementsdemonstrated in Embodiment 1, a second primer having a correlation of aforward primer/reverse primer with this allele specific primer, DNApolymerase for PCR, dNTPs, Mg ion, a buffer, and a desired salt asneeded.

Consequently, when the base corresponding to SNP positioned at the 3′end of the allele specific primer is complementary to the target SNPbase, template DNA in the region sandwiched between this allele specificprimer and the second primer can be efficiency amplified. To thecontrary, when the base corresponding to SNP positioned at the 3′ end ofthe allele specific primer is noncomplementary to the target SNP base,template DNA in the region sandwiched between this allele specificprimer and the second primer is hardly amplified. Accordingly, SNPdiscrimination is enabled which is accompanied by less possibility ofthe false positive and is definite through determination of the amountof amplification of this template DNA by a method such aselectrophoresis following the PCR reaction.

Alternatively, the amount of pyrophosphate released as a result of thePCR reaction may be measured during or following the PCR reaction asdescribed above. Also in this case, SNP discrimination is enabled whichis accompanied by less possibility of the false positive and isdefinite.

The DNA polymerase for PCR described above preferably has week or no3′→5′ exonuclease activity.

Method of measuring the amount of amplification of the template DNA isnot particularly limited, but a method other than the aforementionedelectrophoretic method is permitted.

Examples of the method of measuring the amount of pyrophosphate includethose proposed in Nucleic Acids Research, 2001, Vol. 29, No. 19 e93, WO03/078655 A1, Japanese Patent Provisional Publication No. 2004-141158and the like. However, the method is not limited thereto, but any methodcapable of measuring pyrophosphate is permitted.

Embodiment 4

In this Embodiment 4, a kit for discriminating SNP including the allelespecific primer according to the present invention will be explained.

Users may carry out a primer extension reaction and a reaction formeasuring pyrophosphate using the kit for discriminating SNP explainedin this section, thereby enabling discrimination of the base type at thetarget SNP in each sample with low possibility of the false positive andin an accurate manner.

The kit for discriminating SNP according to this Embodiment 4 comprisesan allele specific primer reagent tube including the allele specificprimer according to the present invention as demonstrated in Embodiment1 for the target DNA sequence, a DNA polymerase reagent tube includingDNA polymerase, a buffer reagent tube including a buffer to which adesired salt is added as needed, a dNTPs reagent tube including dNTPs,and a pyrophosphate reagent tube including a reagent for measuringpyrophosphate.

Therefore, the users first prepare a single stranded DNA or doublestranded DNA sample including each target DNA sequence to give the stateto permit the primer extension reaction to be carried out, andthereafter may carry out the primer extension reaction according to themethod explained in Embodiment 2 using the allele specific primerincluded in the allele specific primer reagent tube, the DNA polymeraseincluded in the DNA polymerase reagent tube, the buffer included in thebuffer reagent tube, and dNTPs included in the dNTPs reagent tube.

Pyrophosphate released into the reaction mixture during the process ofthe primer extension reaction may be measured using a reagent formeasuring pyrophosphate included in the aforementioned pyrophosphatereagent tube.

Accordingly, discrimination of the base type at the target SNP isenabled with low possibility of the false positive and in an accuratemanner.

The reagents for measuring pyrophosphate are not particularly limited aslong as pyrophosphate can be measured, but ATP sulfurylase forconverting pyrophosphate into ATP, luciferin for executing aluminescence reaction (wavelength: 520 nm) through reacting with ATP,and luciferase that catalyzes the reaction may be included, when aluminescence reaction by means of luciferase is used as demonstrated inNucleic Acids Research, 2001, Vol. 29, No. 19 e93. These may be in thestate of being admixed in one tube, or they may be included in separatetubes, respectively. In any case, the users can optically measurepyrophosphate released in the process of the aforementioned primerextension reaction using these reagents for measuring pyrophosphateaccording to the method disclosed in Nucleic Acids Research, 2001, Vol.29, No. 19 e93.

Additionally, in this instance, because dATP remained unreacted in theprimer extension reaction reacts with luciferin, a tube including anenzyme for decomposing the unreacted dATP may preferably be included ina part of the kit. It is preferred that this enzyme is added to theprimer extension reaction liquid before the reaction with luciferase asdescribed above, or before the reaction for converting pyrophosphateinto ATP, thereby executing the dATP decomposing reaction.

With respect to the reagent for measuring pyrophosphate, when theelectrochemical measurement method of pyrophosphate illustrated in WO03/078655A1 is used, pyrophosphatase for hydrolyzing pyrophosphate intophosphoric acid, GAP (glyceraldehyde 3-phosphate) and NAD that reactwith this phosphoric acid, GAPDH that catalyzes the reaction of thisphosphoric acid-GAP-NAD (glyceraldehyde 3-phosphate dehydrogenase), anelectron-transfer mediator (oxidized form) that reacts with NADHobtained by this reaction of phosphoric acid-GAP-NAD, and diaphorasethat catalyzes the reaction of the NADH and the electron-transfermediator may be included. These may be in the state of being admixed inone tube, or they may be included in separate tubes, respectively. Inany case, the users can electrochemically measure pyrophosphate releasedin the process of the aforementioned primer extension reaction using thereagents for measuring pyrophosphate according to the method disclosedin WO 03/078655A1.

With respect to the reagent for measuring pyrophosphate, when theelectrochemical measurement method illustrated in Japanese PatentProvisional Publication No. 2004-141158 is used, a membrane comprisingH⁺-PPase (H⁺-pyrophosphatase) that hydrolyzes pyrophosphate intophosphoric acid and transports H⁺, and a dye stuff that exhibits alteredoptical characteristics depending on the H⁺ concentration may beincluded. These may be in the state of being admixed in one tube, or maybe included in separate tubes, respectively. In any case, the users canoptically measure pyrophosphate released in the process of theaforementioned primer extension reaction using these reagents formeasuring pyrophosphate according to the method disclosed in JapanesePatent Provisional Publication No. 2004-141158.

In connection with the method of measuring pyrophosphate, a number ofmethods other than those demonstrated herein have been known. Therefore,as a matter of course, the reagents for measuring pyrophosphate are notlimited to those demonstrated herein, but any constitution may bepermitted which includes all or a part of reagents required for themeasurement of pyrophosphate.

In stead of the reagent for measuring pyrophosphate, a constitutionincluding a fluorescence indicator that binds to a double stranded DNAsuch as SYBR^((R)) Green I in the kit of this Embodiment 4 may bepermitted.

More specifically, when the primer extension reaction is allowed in thepresence of a fluorescence indicator that binds to a double stranded DNAtypified by SYBR^((R)) Green I, process of the primer extension reactioncan be analyzed in real time by monitoring the fluorescence. SNPdiscrimination can be performed based on the results of this analysis.

The fluorescence indicator that binds to a double stranded DNA typifiedby SYBR^((R)) Green I may be included in one tube alone, or may beconstituted to be included in the buffer reagent tube or the like.Accordingly, the kit may be constituted to permit this fluorescenceindicator to be mixed in the primer extension reaction liquid.

Similarly, in stead of the reagent for measuring pyrophosphate, aconstitution including Taq Man probe in the kit of this Embodiment 4 maybe permitted. In other words, when Taq Man probe is designed to bind toa desired region in the sequence where the primer extension reactionproceeds, process of the primer extension reaction can be analyzed inreal time by monitoring the fluorescence depending on this Taq Manprobe. Accordingly, SNP discrimination can be performed based on theresults of this analysis.

The Taq Man probe designed in such a manner may be included in one tubealone, or may be constituted to be included in the buffer reagent tubeor the like. Thus, the kit may be constituted to permit the Taq Manprobe to be mixed in a primer extension reaction liquid.

The aforementioned DNA polymerase herein preferably has week or no 3′→5′exonuclease activity for achieving more accurate SNP discrimination. Inaddition, it is more preferred that the DNA polymerase is heatresistant.

Embodiment 5

In this Embodiment 5, a kit for discriminating SNP including the allelespecific primer according to the present invention will be explained.

Users may carry out a PCR reaction using the kit for discriminating SNPexplained in this section, thereby enabling discrimination of the basetype at the target SNP in each sample with low possibility of the falsepositive and in an accurate manner.

The kit for discriminating SNP according to this Embodiment 5 comprisesa primer reagent tube including the allele specific primer according tothe present invention as demonstrated in Embodiment 1 for the target DNAsequence and a second primer having a correlation of a forwardprimer/reverse primer with this allele specific primer, a DNA polymerasereagent for PCR tube including DNA polymerase for PCR, a buffer reagenttube including a buffer to which Mg ion and a desired salt are added asneeded, and a dNTPs reagent tube including dNTPs.

Therefore, the users first prepare a double stranded DNA sampleincluding each target DNA sequence to give the state to permit the PCRreaction to be carried out, and thereafter may carry out the PCRreaction according to the method explained in Embodiment 3 using theallele specific primer and the second primer included in the primerreagent tube, the DNA polymerase for PCR included in the DNA polymerasereagent for PCR tube, the buffer included in the buffer reagent tube,and dNTPs included in the dNTPs reagent tube.

It is more preferred that the kit according to this Embodiment 5includes a reagent for examining efficiency of the PCR reaction.Examples of the reagent for examining the efficiency of the PCR reactioninclude, similarly to Embodiment 3, the reagents for measuringpyrophosphate, fluorescence indicators that bind to a double strandedDNA such as SYBR^((R)) Green I, and Taq Man probe.

The reagent for measuring pyrophosphate can measure pyrophosphatereleased into the reaction liquid during the PCR reaction. Therefore,based on the measurement results, discrimination of the base type at thetarget SNP is enabled with low possibility of the false positive and inan accurate manner. Types of the reagent for measuring pyrophosphate aresimilar to those in Embodiment 4, which may be constituted to includeall or a part of the reagents required for the measurement ofpyrophosphate.

In connection with the fluorescence indicator that binds to a doublestranded DNA such as SYBR^((R)) Green I, or Taq Man probe, the processof the PCR reaction can be monitored in real time by carrying out thePCR reaction in the presence of the same. Thus, discrimination of thebase type at the target SNP is enabled with low possibility of the falsepositive and in an accurate manner based on the results.

Therefore, these may be included in one tube alone, or may beconstituted to be included in the buffer reagent tube or the like.Accordingly, the kit may be constituted to permit these reagents to bemixed in the PCR reaction liquid.

The allele specific primer according to the present invention, methodsof discriminating SNP using this allele specific primer, kits fordiscriminating SNP including this allele specific primer were explainedin the foregoings by way of Embodiments 1 to 5.

These are not particularly limited to SNP, but exactly similar effectsmay be achieved as a matter of course, also with respect todiscrimination of difference in single base due to mutation, ordiscrimination of difference in single base caused by artificialmutation at a certain particular base.

EXAMPLES

More specific Examples of the present invention will be described below.

Example 1

In this Example 1, discrimination of the target base type in λDNA wascarried out by a primer extension reaction using the allele specificprimer according to the present invention.

More specifically, a mutant type λDNA-6451-GC was first produced byartificially substituting an A/T base pair at position 6451 in λDNA(manufactured by Takara Bio Inc.) to a G/C base pair.

Then discrimination of substitution of the base pair was performed usingtwo primers of: 5′-GAATCTGGCGGACCCGGCACA-3′ (λ-ACA), and5′-GAATCTGGCGGACCCGGCCCA-3′ (λ-CCA).

The above three primers are referred to as λ-ACA and λ-CCA in thisorder.

The 3′ end base A of the above three primers is complementary to the 5′end base T of 5′-TAAGCCGGGTCCGCCAGATTC-3′ among double stranded DNAsequences of 5′-GAATCTGGCGGACCCGGCTTA-3′/5′-TAAGCCGGGTCCGCCAGATTC-3′consisting of positions 6431 to 6451 of the wild type λDNA.

The second base C from the 3′ end of the above three primers is in anoncomplementary relation of C/A, to the second base A from the 5′ endof 5′-TAAGCCGGGTCCGCCAGATTC-3′, similarly.

The third base types from the 3′ end of the above two primers aredifferent (A in λ-ACA and C in λ-CCA), but they are in anoncomplementary relation, to the third base A from the 5′ end of5′-TAAGCCGGGTCCGCCAGATTC-3′, similarly.

The sequence of from the fourth base from the 3′ end to the 5′ end baseof the above two primers is completely complementary, to sequence offrom the fourth base from the 5′ end to the 3′ end base of5′-TAAGCCGGGTCCGCCAGATTC-3′, similarly.

On the other hand, three bases at the 3′ end of the above two primersare noncomplementary to three bases at the 5′ end of5′-CAAGCCGGGTCCGCCAGATTC-3′ among the double stranded DNA sequences of5′-GAATCTGGCGGACCCGGCTTG-3′/5′-CAAGCCGGGTCCGCCAGATTC-3′ consisting ofpositions 6431 to 6451 of the mutant type λDNA-6451-GC (mutationintroduced site underlined).

The experiment was conducted as follows.

First, 10 μL of a reaction solution was prepared containing:

20 μM wild type λDNA or mutant type λDNA-6451-GC described above,

10 μM of any one of λ-ACA or λ-CCA,

0.5 U/μL TaKaRa Taq (manufactured by Takara Bio Inc.),

10 mM Tris-HCl (pH 8.3),

50 mM KCl,

1.5 mM MgCl₂, and

each 0.25 mM dNTPs.

This reaction solution was incubated using DNA engine that is a thermalcycler manufactured by MJ Research Inc., at 95° C. for 1 minute, thenincubated at 58° C. for 5 minutes, and finally incubated at 72° C. for10 minutes.

The reaction liquid following incubation was analyzed on the amount ofincluded pyrophosphate utilizing a luciferase reaction system. Morespecifically, the amount of pyrophosphate was analyzed throughconverting the amount of pyrophosphate into luminescence intensityaccording to the method of Kambara et el., (Nucleic Acids Research,2001, Vol. 29, No. 19 e93).

Results of the analysis are illustrated in FIG. 5.

These results are shown by comparative luminescence intensity in eachcase when luminescence intensity was assumed to be 100% in case wherethe primer extension reaction was carried out with λ-ACA on the wildtype λDNA.

FIG. 5 reveals that any one of the aforementioned λ-ACA and λ-CCAapparently exhibited greater luminescence intensity in case of the wildtype λDNA compared to the case of the mutant type λDNA-6451-GC.

Accordingly, it is concluded that difference in single base in thisExample could be definitely discriminated by the aforementioned λ-ACAand λ-CCA.

Comparative Example 1

In this Comparative Example 1, discrimination was attempted as acomparative experiment of the above Example 1 on difference in singlebase of the wild type λDNA of the above Example 1 and the mutant typeλDNA-6451-GC using the following two primers.

More specifically, similar experiment to Example 1 was conducted usingtwo primers consisting of the sequence of 5′-GAATCTGGCGGACCCGGCAAA-3′(λ-AAA), and 5′-GAATCTGGCGGACCCGGCGAA-3′ (λ-GAA).

These two primers are referred to as λ-AAA and λ-GAA in this order.

In these sequences, similarly to λ-ACA and λ-CCA used in Example 1, thesecond and the third bases from the 3′ end of these sequences arenoncomplementary to the second and the third bases from the 5′ end of5′-TAAGCCGGGTCCGCCAGATTC-3′.

Results of the analysis are illustrated in FIG. 6.

These results are shown by comparative luminescence intensity in eachcase when luminescence intensity was assumed to be 100% in case wherethe primer extension reaction was carried out with λ-ACA on the wildtype λDNA, similarly to in FIG. 5.

Luminescence intensity in case of combination of these two primers andthe wild type λDNA is apparently smaller compared to the case ofcombination of λ-ACA or λ-CCA and the wild type λDNA (note the magnitudeof ordinate in FIG. 5 and FIG. 6). Accordingly, it is revealed thatdiscrimination capability of λ-ACA and λ-CCA is more excellent.

As is seen from these Example 1 and Comparative Example 1, extremelyhigh discrimination capability can be attained when a base immediatelyadjacent to on the 3′ end side of the target SNP base is A; a baseadjacent with one base spaced apart is A; the second base from the 3′end of the allele specific primer is C; and the third base is any one ofA or C.

Example 2

In this Example 2, discrimination of the target base type in λDNA wascarried out by a PCR reaction using the allele specific primer accordingto the present invention.

A mutant type λDNA-6504-AT was first produced by artificiallysubstituting a G/C base pair at position 6504 in λDNA (manufactured byTakara Bio Inc.) to an A/T base pair.

Then discrimination of substitution of the base pair was performed by aPCR reaction using two primers of: 5′-GCGTGACGAAGAGCTGGCCAACG-3′(λ-ACG), and 5′-GCGTGACGAAGAGCTGGCCACCG-3′ (λ-CCG)as a forward primer.

The above two primers are referred to as λ-ACG and λ-CCG in this order.

The 3′ end base G of the above two primers is complementary to the 5′end base C of 5′-CAATGGCCAGCTCTTCGTCACGC-3′ among the double strandedDNA sequences of5′-GCGTGACGAAGAGCTGGCCATTG-3′/5′-CAATGGCCAGCTCTTCGTCACGC-3′ consistingof positions 6482 to 6504 of the wild type λDNA.

The second base C from the 3′ end of the above two primers is in anoncomplementary relation of C/A, to the second base A from the 5′ endof 5′-CAATGGCCAGCTCTTCGTCACGC-3′, similarly.

The third base types from the 3′ end of the above two primers aredifferent (A in λ-ACG, and C in λ-CCG), but they are in anoncomplementary relation, similarly, to the third base A from the 5′end of 5′-CAATGGCCAGCTCTTCGTCACGC-3′, similarly.

The sequence of from the fourth base from the 3′ end to the 5′ end baseof the above two primers is completely complementary, similarly, tosequence of from the fourth base from the 5′ end to the 3′ end base of5′-CAATGGCCAGCTCTTCGTCACGC-3′.

On the other hand, three bases at the 3′ end of the above three primersare noncomplementary to three bases at the 5′ end of5′-TAATGGCCAGCTCTTCGTCACGC-3′ among the double stranded DNA sequences of5′-GCGTGACGAAGAGCTGGCCATTA-3′/5′-TAATGGCCAGCTCTTCGTCACGC-3′ consistingof positions 6482 to 6504 of the mutant type λDNA-6504-AT (mutationintroduced site underlined).

As a reverse primer, a DNA consisting of 5′-GAATCACGGTATCCGGCTGCGCTGA-3′was used. This reverse primer is completely complementary to5′-TCAGCGCAGCCGGATACCGTGATTC-3′ among the double stranded DNA sequencesof 5′-TCAGCGCAGCCGGATACCGTGATTC-3′/5′-GAATCACGGTATCCGGCTGCGCTGA-3′consisting of positions 7406 to 7430 of the λDNA.

Provided that a PCR reaction with the aforementioned λ-ACG or λ-CCGfavorably proceeds, a DNA amplification product of 949 bp will beobtained.

The experiment was conducted as follows.

First, 20 μL of a reaction solution was prepared containing:

2 μL of an enzyme mixture of Light Cycler-FastStart DNA Master SYBRGreen I kit (manufactured by Roche Diagnostics),

10 μg/mL wild type λDNA or mutant type λDNA-6504-AT described above,

1 μM of any one of λ-ACG or λ-CCG,

1 μM reverse primer described above, and

1.6 mM MgCl₂.

This reaction solution was subjected to a PCR reaction using LightCyclerthat is a thermal cycler manufactured by Roche Diagnostics, under thecondition of denaturation step: 94° C., 10 sec; annealing step : 58° C.,10 sec; extension step 72° C., 10 sec; and cycle number: 20 cycles.

Results of each PCR reaction were analyzed using Bioanalyzer 2100 thatis a system for DNA electrophoresis manufactured by Agilent TechnologiesInc.

Results of the analysis are illustrated in FIG. 7.

These results are shown by concentration (nM) of the target DNA fragmentfollowing each PCR reaction.

According to these results, in every case where any one of theaforementioned λ-ACG or λ-CCG was used, the target DNA fragment was notdetected in case of the mutant type λDNA-6504-AT.

To the contrary, in every case of the wild type λDNA, concentration ofthe target DNA fragment was equal to or greater than 20 nM. Accordingly,it is concluded that the difference in single base in this Example couldbe definitely discriminated by carrying out a PCR reaction using theaforementioned λ-ACG or λ-CCG as a forward primer.

Comparative Example 2

In this Comparative Example 2, discrimination was attempted as acomparative experiment of the above Example 2 on difference in singlebase of the wild type λDNA of the above Example 2 and the mutant typeλDNA-6504-AT using the following two primers.

More specifically, similar experiment to Example 2 was conducted usingtwo primers consisting of the sequence of 5′-GCGTGACGAAGAGCTGGCCAAAG-3′(λ-AAG) and 5′-GCGTGACGAAGAGCTGGCCAGAG-3′ (λ-GAG).

These two primers are referred to as λ-AAG and λ-GAG in this order.

In these sequences, similarly to λ-ACG and λ-CCG used in Example 2, thesecond and the third bases from the 3′ end of these sequences arenoncomplementary to the second and the third bases from the 5′ end of5′-CAATGGCCAGCTCTTCGTCACGC-3′.

The residual sequence is completely complementary to the residualsequence of 5′-CACGCTTGCTCCACTCCGTGCCGCC-3′.

Results of the analysis are illustrated in FIG. 8.

These results are shown by concentration (nM) of the target DNA fragmentfollowing each PCR reaction, similarly to in FIG. 7.

Thus, as for each of λ-AAG and λ-GAG, the target DNA fragment of equalto or greater than 1.5 nM was detected first although slight in case ofthe wild type λDNA, while any detection could not be resulted in case ofthe mutant type λDNA-6504-AT.

Concentration of the target DNA fragment in cases of combination of anyone of these two primers and the wild type λDNA was less than abouttenth compared to the case of combination of any one of λ-ACG or λ-CCGand the wild type λDNA (note the magnitude of ordinate in FIG. 7 andFIG. 8). Accordingly, it is revealed that discrimination capability ofλ-ACG and λ-CCG is more excellent.

According to the present invention, a method of discriminating a targetbase carried by a DNA which is accompanied by less possibility of thefalse positive and enables definite discrimination of SNP or the like isprovided.

From the foregoing descriptions, many modifications and otherembodiments of the present invention will be apparent to persons skilledin the art. Therefore, the foregoing descriptions should be construed asjust for illustrative exemplification, provided for the purpose ofteaching the best embodiment for carrying out the present invention topersons skilled in the art. Details of construction and/or function ofthe present invention can be substantially altered without departingfrom the spirit thereof.

1. A method of discriminating a target base carried by a DNA, saidmethod which comprises: (1) a DNA to elongation step comprising bindingto said DNA an allele specific primer having at its 3′ end a base thatis complementary to a base predicted as said target base among 4 basesof A, T, G and C to cause a DNA elongation reaction, and (2) adiscrimination step comprising examining efficiency of said DNAelongation to discriminate that said target is the same as saidpredicted base when the efficiency is high, or that said target base isdistinct from said predicted base when the efficiency is low, wherein insaid DNA, a base immediately adjacent to on the 3′ end side of saidtarget base is A, and a base adjacent with one base spaced apart is A,3′ end base of said allele specific primer is complementary to the basepredicted to be said target base, the second base from the 3′ end ofsaid allele specific primer is C, the third base from the 3′ end of saidallele specific primer is any one of A or C, and a base sequence betweenthe fourth base from the 3′ end and the 5′ end base of said allelespecific primer complementarily binds to a base sequence between thebase on said DNA corresponding to the fourth base from the 3′ end ofsaid allele specific primer and the base on said DNA corresponding tothe 5′ end base of said allele specific primer.
 2. The method accordingto claim 1 wherein said DNA elongation reaction is a primer extensionreaction.
 3. The method according to claim 1 wherein said DNA elongationreaction is a primer extension reaction relied solely on said allelespecific primer.
 4. The method according to claim 2 wherein saidefficiency is examined by determining the concentration of pyrophosphateproduced by said primer extension reaction.
 5. The method according toclaim 4 wherein said concentration of pyrophosphate is detected in termsof luminescence intensity.
 6. The method according to claim 1 whereinsaid DNA elongation reaction is a PCR reaction.
 7. The method accordingto claim 1 wherein said DNA elongation reaction is a PCR reaction reliedon a combination of said allele specific primer with the other differentprimer.
 8. The method according to claim 6 wherein said efficiency isexamined by measuring the concentration of the amplified DNA produced bysaid PCR reaction with an electrophoretic method.
 9. (canceled)